PROJECT SUMMARY/ABSTRACT: This K08 Career Development Award details a five-year training program to advance Dr. Lindsey George?s career goal of becoming an independent physician-scientist translating biochemical and molecular studies to develop novel therapeutics for patients with disorders of blood coagulation, specifically hemophilia. During the award period, Dr. George will continue developing her expertise in coagulation biochemistry and gene therapy, acquire new scientific skills in mouse modeling, viral vector development and advance her translational capabilities. Under the guidance of her mentor, Dr. Rodney Camire, these training objectives will be met by a combination of didactic course work, participation in seminar series, research experience, and mentoring by her advisory committee. Her advisory committee is composed of world renowned scientists with extensive mentoring experience and diverse and complementary scientific expertise including Drs. Lawrence Brass, Mortimer Poncz, and Beverly Davidson. The scientific proposal is aimed at addressing the current limitations of therapies for hemophilia A. Hemophilia A is due to an inheritable deficiency in coagulation (F)actor VIII activity. The current standard-of-care for hemophilia A is enzyme replacement therapy with intravenous FVIII infusions. There are several novel therapeutics in various phases of clinical development that are poised to alter the current treatment paradigm, but require mechanistic understanding to ensure safety and maximal efficacy in clinical adaptation. Specifically, important knowledge gaps in the field relate to mechanisms by which FVIII is downregulated. This has direct clinical relevance to understanding the safety and/or efficacy of FVIII-mimetic bispecific antibodies that bypass natural FVIII regulatory pathways and interpreting FVIII gene therapy data wherein ectopic hepatocyte expression may alter FVIII biology. Mechanisms regulating FVIIIa inactivation are thought to be rapid spontaneous dissociation of the non-covalently bound FVIIIa-A2 subunit and proteolytic processing by activated protein C; however, basic and clinical observations suggest that activated protein C has minimal physiologic consequence in FVIIIa regulation. Dr. George?s proposal focuses on determining the mechanism of FVIIIa inactivation and translating this understanding for purposes of improving and developing novel therapies for hemophilia A. She has developed unique reagents and a rational approach to this timely, mechanistic question. In Aim 1, the physiologic relevant mechanism of FVIIIa inactivation will be determined by in vitro purified systems assays and in vivo investigation. In Aim 2, the mechanism of FVIIIa inactivation will be exploited for therapeutic translation. Specifically, Dr. George will determine if there are differences in FVIIIa inactivation among native endothelial versus hepatocyte-derived FVIII. She will additionally apply information learned from Aim 1 to rationally design FVIII variants with impaired inactivation and determine their effect on hemostasis in the setting of both gene transfer and recombinant protein through in vitro kinetic studies and in vivo gene transfer studies. !